It has been previously demonstrated that the enzymic activity of rat liver, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), is modulated in vitro and in vivo by a bicyclic cascade system involving reversible phosphorylation of HMGR and reductase kinase (RK). Administration of mevalonolactone (MVL) (600 mg/kg) to rats caused a rapid biphasic inhibition of HMGR. The first phase (20 min) of reversible inhibition (30%) was due to enhanced HMGR phosphorylation. The second phase (60 min) of MVL inhibition of HMGR activity (80%) was an irreversible process, not involving a decrease in quantity of purified HMGR protein in comparision to controls. These data suggest the presence of an additional new mechanism for the regulation of HMGR activity. The increase in HMGR phosphorylation (20 min) was associated with an increased phosphorylation and activation (2-3 fold) of both cytosolic and microsomal RK. Increased activation (phosphorylation) of RK was catalyzed by reductase kinase, kinase (RKK) which was significantly activated (2-3 fold) following the administration of MVL. The mechanism of the in vivo activation of RKK is not yet known. Both RK and RKK activities remained elevated for 60 min after RVL administration. In addition, a relative increase in the degree of phosphorylation of HMGR and RK occured due to a significant decrease in phosphatase activity. A 35% and 54% reduction in phosphatase activity (expressed in terms of HMGR dephosphorylation) occured 20 and 60 min after MVL administration respectively. These combined data indicate that MVL mediated short-term in vivo inhibition of HMGR activity occured by two mechanisms: 1) an increase in the degree of phosphorylation of both HMGR and RK due to increased activities of RK and RKK; 2) a decrease in the dephosphorylation of both HMGR and RK due to a reduction in the phosphatase activity. These combined effects would favor an increase in the steady state level of the phosphorylated forms of both HMGR and RK resulting in a reduction in the enzymic activity of HMGR. These results also represent the initial demonstration that the activity of RKK is modulated in vivo providing a mechanism for the regulation of the activities of both RK and HMGR.